Hypothesis for heritable, anti-viral immunity in crustaceans and insects

Correction to Flegel, TW: Hypothesis for heritable, anti-viral immunity in crustaceans and insects. Biology Direct 2009, 4:32

Response to Dengue virus infections altered by cytokine-like substances from mosquito cell cultures

<p>Abstract</p> <p>Background</p> <p>With both shrimp and commercial insects such as honey bees, it is known that stable, persistent viral infections characterized by absence of disease can sometimes shift to overt disease states as a result of various stress triggers and that this can result in serious economic losses. The main research interest of our group is to understand the dynamics of stable viral infections in shrimp and how they can be destabilized by stress. Since there are no continuous cell lines for crustaceans, we have used a C6/36 mosquito cell line infected with Dengue virus to test hypotheses regarding these interactions. As a result, we accidentally discovered two new cytokine-like substances in 5 kDa extracts from supernatant solutions of acutely and persistently infected mosquito cells.</p> <p>Results</p> <p>Naïve C6/36 cells were exposed for 48 h to 5 kDa membrane filtrates prepared from the supernatant medium of stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Subsequent challenge of naïve cells with a virulent stock of Dengue virus 2 (DEN-2) and analysis by confocal immunofluorescence microscopy using anti-DEN-2 antibody revealed a dramatic reduction in the percentage of DEN-2 infected cells when compared to control cells. Similar filtrates prepared from C6/36 cells with acute DEN-2 infections were used to treat stable C6/36 mosquito cell cultures persistently-infected with Dengue virus. Confocal immunofluorescence microscopy revealed destabilization in the form of an apoptosis-like response. Proteinase K treatment removed the cell-altering activities indicating that they were caused by small polypeptides similar to those previously reported from insects.</p> <p>Conclusions</p> <p>This is the first report of cytokine-like substances that can alter the responses of mosquito cells to Dengue virus. This simple model system allows detailed molecular studies on insect cytokine production and on cytokine activity in a standard insect cell line.</p

False rumours of disease outbreaks caused by infectious myonecrosis virus (IMNV) in the whiteleg shrimp in Asia

<p>Abstract</p> <p>Background</p> <p>Infectious myonecrosis virus (IMNV) disease outbreaks in cultivated whiteleg shrimp <it>Penaeus (Litopenaeus) vannamei </it>are characterized by gross signs of whitened abdominal muscles and by slow mortality reaching up to 70%. In 2006 the first disease outbreaks caused by IMNV in Asia occurred in Indonesia. Since then rumours have periodically circulated about IMNV disease outbreaks in other Asian countries. Our findings indicate that these are false rumours.</p> <p>Findings</p> <p>Our continual testing by nested RT-PCR of shrimp samples suspected of IMNV infection from various Asian countries since 2006 has yielded negative results, except for samples from Indonesia. Our results are supported by the lack of official reports of IMNV outbreaks since January 2007 in the Quarterly Report on Aquatic Animal Diseases (QAAD) from the Network of Aquaculture Centers in Asia Pacific (NACA). In most cases, our shrimp samples for which tissue sections were possible showed signs of muscle cramp syndrome that also commonly causes muscle whitening in stressed whiteleg shrimp. Thus, we suspect that most of the false rumours in Asia about IMNV outside of Indonesia have resulted because of muscle cramp syndrome.</p> <p>Conclusions</p> <p>Results from continual testing of suspected IMNV outbreaks in Asian countries other than Indonesia since 2006 and the lack of official country reports of IMNV outbreaks since January 2007, indicate that rumours of IMNV outbreaks in Asian countries outside of Indonesia are false. We suspect that confusion has arisen because muscle cramp syndrome causes similar signs of whitened tail muscles in whiteleg shrimp.</p

Aerodynamic Effects of High Turbulence Intensity on a Variable-Speed Power-Turbine Blade With Large Incidence and Reynolds Number Variations

The effects of high inlet turbulence intensity on the aerodynamic performance of a variable speed power turbine blade are examined over large incidence and Reynolds number ranges. These results are compared to previous measurements made in a low turbulence environment. Both high and low turbulence studies were conducted in the NASA Glenn Research Center Transonic Turbine Blade Cascade Facility. The purpose of the low inlet turbulence study was to examine the transitional flow effects that are anticipated at cruise Reynolds numbers. The current study extends this to LPT-relevant turbulence levels while perhaps sacrificing transitional flow effects. Assessing the effects of turbulence at these large incidence and Reynolds number variations complements the existing database. Downstream total pressure and exit angle data were acquired for 10 incidence angles ranging from +15.8deg to 51.0deg. For each incidence angle, data were obtained at five flow conditions with the exit Reynolds number ranging from 2.1210(exp 5) to 2.1210(exp 6) and at a design exit Mach number of 0.72. In order to achieve the lowest Reynolds number, the exit Mach number was reduced to 0.35 due to facility constraints. The inlet turbulence intensity, Tu, was measured using a single-wire hotwire located 0.415 axial-chord upstream of the blade row. The inlet turbulence levels ranged from 8 to 15 percent for the current study. Tu measurements were also made farther upstream so that turbulence decay rates could be calculated as needed for computational inlet boundary conditions. Downstream flow field measurements were obtained using a pneumatic five-hole pitch/yaw probe located in a survey plane 7 percent axial chord aft of the blade trailing edge and covering three blade passages. Blade and endwall static pressures were acquired for each flow condition as well. The blade loading data show that the suction surface separation that was evident at many of the low Tu conditions has been eliminated. At the extreme positive and negative incidence angles, the data show substantial differences in the exit flow field. These differences are attributable to both the higher inlet Tu directly and to the thinner inlet endwall boundary layer that the turbulence grid imposes

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp

<p>Abstract</p> <p>Background</p> <p>From 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species.</p> <p>Results</p> <p>Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number <ext-link ext-link-id="FJ498866" ext-link-type="gen">FJ498866</ext-link>). <it>In situ </it>hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and <it>in situ </it>hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM.</p> <p>Conclusions</p> <p>ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp.</p

Aerodynamic Measurements of a Variable-Speed Power-Turbine Blade Section in a Transonic Turbine Cascade at Low Inlet Turbulence

Aerodynamic measurements obtained in a transonic linear cascade were used to assess the impact of large incidence angle and Reynolds number variations on the 3-D flow field and midspan loss and turning of a 2-D section of a variable-speed power-turbine (VSPT) rotor blade. Steady-state data were obtained for ten incidence angles ranging from +15.8 deg to 51.0 deg. At each angle, data were acquired at five flow conditions with the exit Reynolds number (based on axial chord) varying over an order-of-magnitude from 2.1210(exp 5) to 2.1210(exp 6). Data were obtained at the design exit Mach number of 0.72 and at a reduced exit Mach number of 0.35 as required to achieve the lowest Reynolds number. Midspan total-pressure and exit flow angle data were acquired using a five-hole pitch/yaw probe surveyed on a plane located 7.0 percent axial chord downstream of the blade trailing edge plane. The survey spanned three blade passages. Additionally, three-dimensional half-span flow fields were examined with additional probe survey data acquired at 26 span locations for two key incidence angles of +5.8 deg and 36.7 deg. Survey data near the endwall were acquired with a three-hole boundary-layer probe. The data were integrated to determine average exit total-pressure and flow angle as functions of incidence and flow conditions. The data set also includes blade static pressures measured on four spanwise planes and endwall static pressures. Tests were conducted in the NASA Glenn Transonic Turbine Blade Cascade Facility. The measurements reflect strong secondary flows associated with the high aerodynamic loading levels at large positive incidence angles and an increase in loss levels with decreasing Reynolds number. The secondary flows decrease with negative incidence as the blade becomes unloaded. Transitional flow is admitted in this low inlet turbulence dataset, making it a challenging CFD test case. The dataset will be used to advance understanding of the aerodynamic challenges associated with maintaining efficient power turbine operation over a wide shaft-speed range. de

Persistent, triple-virus co-infections in mosquito cells

<p>Abstract</p> <p>Background</p> <p>It is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.</p> <p>Results</p> <p>Using immunocytochemistry by confocal microscopy, we found that JE super-challenge of cells dually infected with DEN and DNV resulted in stable cultures without signs of cytopathology, and with 99% of the cells producing antigens of the 3 viruses. Location of antigens for all 3 viruses in the triple co-infections was dominant in the cell nuclei. Except for DNV, this differed from the distribution in cells persistently infected with the individual viruses or co-infected with DNV and DEN. The dependence of viral antigen distribution on single infection or co-infection status suggested that host cells underwent an adaptive process to accommodate 2 or more viruses.</p> <p>Conclusions</p> <p>Individual mosquito cells can accommodate at least 3 viruses simultaneously in an adaptive manner. The phenomenon provides an opportunity for genetic exchange between diverse viruses and it may have important medical and veterinary implications for arboviruses.</p

Mud crab susceptibility to disease from white spot syndrome virus is species-dependent

<p>Abstract</p> <p>Background</p> <p>Based on a report for one species (<it>Scylla serrata</it>), it is widely believed that mud crabs are relatively resistant to disease caused by white spot syndrome virus (WSSV). We tested this hypothesis by determining the degree of susceptibility in two species of mud crabs, <it>Scylla olivacea </it>and <it>Scylla paramamosain</it>, both of which were identified by mitochondrial 16 S ribosomal gene analysis. We compared single-dose and serial-dose WSSV challenges on <it>S. olivacea </it>and <it>S. paramamosain</it>.</p> <p>Findings</p> <p>In a preliminary test using <it>S. olivacea </it>alone, a dose of 1 × 10⁶WSSV copies/g gave 100% mortality within 7 days. In a subsequent test, 17 <it>S. olivacea </it>and 13 <it>S. paramamosain </it>were divided into test and control groups for challenge with WSSV at 5 incremental, biweekly doses starting from 1 × 10⁴and ending at 5 × 10⁶copies/g. For 11 <it>S. olivacea </it>challenged, 3 specimens died at doses between 1 × 10⁵and 5 × 10⁵copies/g and none died for 2 weeks after the subsequent dose (1 × 10⁶copies/g) that was lethal within 7 days in the preliminary test. However, after the final challenge on day 56 (5 × 10⁶copies/g), the remaining 7 of 11 <it>S. olivacea </it>(63.64%) died within 2 weeks. There was no mortality in the buffer-injected control crabs. For 9 <it>S. paramamosain </it>challenged in the same way, 5 (55.56%) died after challenge doses between 1 × 10⁴and 5 × 10⁵copies/g, and none died for 2 weeks after the challenge dose of 1 × 10⁶copies/g. After the final challenge (5 × 10⁶copies/g) on day 56, no <it>S. paramamosain </it>died during 2 weeks after the challenge, and 2 of 9 WSSV-infected <it>S. paramamosain </it>(22.22%) remained alive together with the control crabs until the end of the test on day 106. Viral loads in these survivors were low when compared to those in the moribund crabs.</p> <p>Conclusions</p> <p><it>S. olivacea </it>and <it>S. paramamosain </it>show wide variation in response to challenge with WSSV. <it>S. olivacea </it>and <it>S. paramamosain </it>are susceptible to white spot disease, and <it>S. olivacea </it>is more susceptible than <it>S. paramamosain</it>. Based on our single-challenge and serial challenge results, and on previous published work showing that <it>S. serrata </it>is relatively unaffected by WSSV infection, we propose that susceptibility to white spot disease in the genus <it>Scylla </it>is species-dependent and may also be dose-history dependent. In practical terms for shrimp farmers, it means that <it>S. olivacea </it>and <it>S. paramamosain </it>may pose less threat as WSSV carriers than <it>S. serrata</it>. For crab farmers, our results suggest that rearing of <it>S. serrata </it>would be a better choice than <it>S. paramamosain </it>or <it>S. olivacea </it>in terms of avoiding losses from seasonal outbreaks of white spot disease.</p

Co-Interactive DNA-Binding between a Novel, Immunophilin-Like Shrimp Protein and VP15 Nucleocapsid Protein of White Spot Syndrome Virus

White spot syndrome virus (WSSV) is one of the most serious pathogens of penaeid shrimp. Although its genome has been completely characterized, the functions of most of its putative proteins are not yet known. It has been suggested that the major nucleocapsid protein VP15 is involved in packaging of the WSSV genome during virion formation. However, little is known in its relationship with shrimp host cells. Using the yeast two-hybrid approach to screen a shrimp lymphoid organ (LO) cDNA library for proteins that might interact with VP15, a protein named PmFKBP46 was identified. It had high sequence similarity to a 46 kDa-immunophilin called FKBP46 from the lepidopteran Spodoptera frugiperda (the fall armyworm). The full length PmFKBP46 consisted of a 1,257-nucleotide open reading frame with a deduced amino acid sequence of 418 residues containing a putative FKBP-PPIase domain in the C-terminal region. Results from a GST pull-down assay and histological co-localization revealed that VP15 physically interacted with PmFKBP46 and that both proteins shared the same subcellular location in the nucleus. An electrophoretic mobility shift assay indicated that PmFKBP46 possessed DNA-binding activity and functionally co-interacted with VP15 in DNA binding. The overall results suggested that host PmFKBP46 might be involved in genome packaging by viral VP15 during virion assembly

Complementary Aerodynamic Performance Datasets for Variable Speed Power Turbine Blade Section from Two Independent Transonic Turbine Cascades

Two independent experimental studies were conducted in linear cascades on a scaled, two-dimensional mid-span section of a representative Variable Speed Power Turbine (VSPT) blade. The purpose of these studies was to assess the aerodynamic performance of the VSPT blade over large Reynolds number and incidence angle ranges. The influence of inlet turbulence intensity was also investigated. The tests were carried out in the NASA Glenn Research Center Transonic Turbine Blade Cascade Facility and at the University of North Dakota (UND) High Speed Compressible Flow Wind Tunnel Facility. A large database was developed by acquiring total pressure and exit angle surveys and blade loading data for ten incidence angles ranging from +15.8deg to 51.0deg. Data were acquired over six flow conditions with exit isentropic Reynolds number ranging from 0.05106 to 2.12106 and at exit Mach numbers of 0.72 (design) and 0.35. Flow conditions were examined within the respective facility constraints. The survey data were integrated to determine average exit total-pressure and flow angle. UND also acquired blade surface heat transfer data at two flow conditions across the entire incidence angle range aimed at quantifying transitional flow behavior on the blade. Comparisons of the aerodynamic datasets were made for three "match point" conditions. The blade loading data at the match point conditions show good agreement between the facilities. This report shows comparisons of other data and highlights the unique contributions of the two facilities. The datasets are being used to advance understanding of the aerodynamic challenges associated with maintaining efficient power turbine operation over a wide shaft-speed range